Diabetes mellitus is a metabolic disorder characterized by the failure of body tissues to oxidize carbohydrates at the normal rate. A deficiency of insulin is the most important factor in the diabetic disease state. During the last 70 years people suffering from diabetes have been greatly aided by receiving controlled amounts of insulin. Until the early 1980's, the insulin used by diabetics was isolated from animal pancreases, generally bovine and porcine. With the introduction of recombinant DNA technology it has become possible to produce large amounts of natural human insulin as well as naturally and non-naturally occurring analogs of human insulin using known synthetic methods. The analogs can also be chemically synthesized. These insulin analogs display different physical and chemical properties when compared to natural human insulin. One such property is to decrease the rate of action of the insulin so that the dosage can be metered into the patient over time as a result of implanting a single dosage.
The human insulin monomer is composed of two chains, the 21 amino acid A chain and the 30 amino acid B chain, that are covalently attached by two interchain disulfide bonds (Merck Index (11th Edition, 789-790 (1989)). In the presence of Zn, natural human insulin associates to a hexamer with 2 Zn atoms coordinated octahedrally to H.sup.B10 of each monomer and 3 water molecules (Blundell, et al., Adv. Protein Chem., 26, 279-402 (1972); and Baker, et al., Philos, Trans. R. Soc. London, B 319, 369-456 (1988)). The 2 Zn insulin hexamer functions as an allosteric protein. Phenolic ligands or certain salts are capable of inducing a conformational transition, resulting in the N-terminal 8 amino acids of the B chain converting from an extended conformation to an Q-helix. The two Zn atoms become tetrahedrally coordinated to H.sup.B10 of each monomer and a fourth solvent-accessible site occupied by small anionic ligands, i.e., C1 ion (Brader & Dunn, TIBS, 16, 341-345 (1991)). This conformational state induced by phenolic ligands has been referred to as the R state and the apoinsulin form as the T state, named after the general nomenclature of Monod et al. (Monod, et al., J. Mol. Biol., 12 88-118 (1965)). The R state is more compact, less flexible, and the Zn exchange is retarded compared to the T state (Derewenda et al., Nature, 338, 594-596 (1989)). The insulin T-&gt;R conversion involves the movement of &gt;30.ANG. of the B.sup.1 .alpha.-carbon (Derewenda et al., Nature, 338, 594-596 (1989)), which is the largest distance transversed by any atom associated with an allosteric conformational transition. A stable intermediate state, T.sub.3 R.sub.3 has been identified that has one trimer in the T state and the other in the R state (Chothia et al., Nature, 302, 500-505 (1983)). The T.sub.3 R.sub.3 state was formally known as the 4-Zn insulin structure which is induced by salts or by limited amounts of phenolics (Kruger, et al., Biol. Chem. Hoppe-Seyler, 371, 669-673 (1990)).
The different allosteric states of insulin hexamer have been best characterized in the crystal state by X-ray crystallography (Bentley et al., Nature, 261, 166-168 (1976); Smith & Dodson, Biopolymers, 32, 441-445 (1992)), in solution by proton NMR, circular dichroism (Renscheidt et al., Eur. J. Biochem., 142, 7-14 (1984), and visible absorption spectroscopy of Co.sup.2+ substituted insulins (Brader et al., Biochemistry, 30, 6636-6645 (1991)). The biological significance of insulin allosterism has not been fully elucidated. The biologically active form of insulin is thought to be a monomer due to the dilute concentrations of insulin in the blood circulation (Frank et al., Diabetes, 21 (Suppl. 2), 486-491 (1972)). A receptor-mediated conformational change in the insulin conformation is thought to be required for binding. A conformational change similar to the T-&gt;R transition, which is induced by the receptor, has been proposed (Derewenda et al., Nature 338, 594-596 (1989)). For the medicinal use of insulin, the T-&gt;R conformations have important consequences. Most formulations of insulin are solutions or suspensions that contain phenolics that function as preservatives against bacterial contamination. The phenolic concentrations in insulin formations are 2-10 times that necessary to induce the R conformation (Kruger et al., Biol. Chem. Hoppe-Seyler, 371, 669-673 (1990)). The presence of phenolics in insulin formations has important consequences on the shelf-life stability (Brange et al., Pharm. Res., 9, 715-726 (1992a); Brange et al., Pharm. Res. 9, 727-734 (1992b); Brange & Langkjaer, Acta Pharm. Nord., 4, 149-158 (1992)) and possibly the time action profile. The solution state storage of insulin has been explained by a thermodynamic model (Brems et al., Protein Engineering, 5, 519-525 (1992)), wherein insulin degradation is governed by the equilibrium constant of unfolding, Keq. The equilibrium constant determined from the reaction N&lt;--&gt;U is U/N, where N=native and U=unfolded. Since the R state of insulin is most compact, least flexible, and the exchange of Zn is retarded, the R state is expected to provide the greatest protection from degradation. Minimizing degradation of insulin formulations is extremely important in reducing undesirable side effects of insulin therapy.
Undoubtedly, millions of patients have benefitted by the serendipitous choice of the original formulators of insulin to use phenolic preservatives. The rate of absorption of insulin from the depot injection site has been shown to be related to the dissociation constant for self-association (Brange, et al., Diabetes Care, 13, 923-954 (1990)). Monomers are thought to be the state that is readily absorbed and the dissociation process to be rate limiting (Binder, C., Artificial Systems for Insulin Delivery, edited by P. Brunetti et al., Raven Press, N.Y., pages 53-57 (1983)). Insulin analogs that are monomeric have been shown to be rapidly absorbed and result in a rapid time action profile (Brange, et al., Curr. Opin. Struct. Biol., 1, 934-940 (1991); DiMarchi et al., Peptides: Chemistry and Biology, Proceedings of the Twelfth American Peptide Symposium, ESCOM, Leiden, pp. 26-28 (1992)). Conversely, insulin forms that have increased association constants should prolong the rate of absorption and the time action profile.
All current intermediate to slow acting insulin formulations are suspensions. When these insulin formulations are injected subcutaneously, they form a depot from which they are slowly absorbed into the blood stream. The dissolution of particles at the subcutaneous injection site is the rate-limiting process that causes delayed time action of these suspension formulations. Suspensions for parenteral use have inherent disadvantages compared to soluble formulations that include poor dosing accuracy, a requirement for resuspension before injection, and a propensity for clumping. A soluble slow-acting insulin formation would overcome these disadvantages and be highly desirable.
Soluble insulin formations demonstrate a lag in their absorption kinetics due to size constraints of the insulin hexamer whose uptake is delayed by dissociation to monomer which is readily absorbed (Brange, J., et al., Current Opinion in Structural Biology, 1, 934-940 (1991)). Various ligands are known to alter the self-association of insulin. As indicated, zinc causes soluble insulin to self-associate to the hexamer (Goldman, J., and Carpenter, F. H., Biochemistry, 13, 4566-4574 (1974)) and hexamers undergo the T-&gt;R transition by the addition of the phenolics (Brader, M. L., et al., TIBS, 16, 341-345 (1991)). The presence of formulation excipients such as Zn and phenols are specific ligands for insulin hexamer and likely delay the dissociation to monomer at the injection site. The binding of phenol is considerably weaker than Zn, and thus the dissolution of insulin hexamer that occurs at the injection site is thought to proceed from the R-state to the T-state and ultimately to monomer.
One strategy for postponing insulin hexamer dislocation is to prevent dissociation using extrinsic ligands such as Zn and phenols. This could be accomplished by creating an insulin analog with the constitutive properties of these extrinsic ligands. The hexamer inducing properties of Zn can be obtained by substituting gluB13 for gluB13 (Bentley, G. A., et al., J. Mol. Biol. 228, 1163-1176 (1992)). The center of the insulin hexamer has six glutamic acids packed closely together causing an electrostatic repulsion and destabilization. By substituting these residues for neutral ones, the insulin analog forms stable hexamers even in the absence of Zn. However, the effect on insulin absorption rate after injection of a acidic solution of the gln.sup.B13 analog is only reduced by 25% (Brange, J., et al., Current Opinion in Structural Biology, 1, 934-940 (1991)). Studies on a mutated insulin by Wollmer et al. (Wollmer et al., Biol. Chem. Hoppe-Seyler, 370, 1045-1053 (1989)) have shown a self-induced allosterism. Glu.sup.B13 insulin plus Zn in the absence of other inducing allosteric ligands has CD spectral properties intermediate between the T and R states (Wollmer et al., Biol. Chem. Hoppe-Seyler, 370, 1045-1053 (1989)). However, experiments were not conducted to distinguish between the existence of a true intermediate T.sub.3 R.sub.3 state or mixture composed of T.sub.6 and R.sub.6 forms (Wollmer et al., Biol. Chem. Hoppe-Seyler, 370, 1045-1053 (1989)).